Environmental Microbiology (2018) 20(1), 16–29 doi:10.1111/1462-2920.

13976

Minireview

Ecological and evolutionary dynamics of a model

facultative pathogen: Agrobacterium and crown gall
disease of plants

Ian S. Barton,1 Clay Fuqua,1 and Thomas G. Platt2* dynamics in disease and non-disease environments
and are subject to shifting selective pressures that
1
Department of Biology, Indiana University, Bloomington, can result in pathoadaptation or the within-host
IN, USA. spread of avirulent phenotypes.
2
Division of Biology, Kansas State University,
Manhattan, KS, USA.

Introduction
The spread of pathogens through host populations
reflects the interplay of many factors including host-
Summary microbe and microbe-microbe interactions as well as
broader epidemiological and evolutionary dynamics
Many important pathogens maintain significant
(Johnson et al., 2015; Parratt et al., 2016). Pathogen life
populations in highly disparate disease and non-
histories vary dramatically and accordingly help deter-
disease environments. The consequences of this
mine which factors are most important to the ecological
environmental heterogeneity in shaping the
and evolutionary dynamics of a particular infectious dis-
ecological and evolutionary dynamics of these
ease. Environmentally acquired infectious agents con-
facultative pathogens are incompletely understood.
tend with the challenge of transitioning between highly
Agrobacterium tumefaciens, the causative agent for
disparate disease and non-disease environments
crown gall disease of plants has proven a productive
(Sokurenko et al., 2006; Brown et al., 2012). Environ-
model for many aspects of interactions between
mentally transmitted pathogens include those that reside
pathogens and their hosts and with other microbes.
within environmental reservoirs in dormant states such
In this review, we highlight how this past work
as spores or virions as well as facultative pathogens
provides valuable context for the use of this system
that are metabolically active in both disease and non-
to examine how heterogeneity and transitions
disease environments.
between disease and non-disease environments
The environmental heterogeneity that these patho-
influence the ecology and evolution of facultative
gens must navigate can be extreme. Host responses
pathogens. We focus on several features common
help shape the within-host success of microbes and
among facultative pathogens, such as the
each environment presents different nutrient availabilities
physiological remodelling required to colonize hosts
and microbial communities. Transitioning between these
from environmental reservoirs and the consequences
environments typically requires that these pathogens
of competition with host and non-host associated
remodel their physiology and behaviour and involves a
microbiota. In addition, we discuss how the life
shift in evolutionary selective pressures acting on the
history of facultative pathogens likely often results in
population. This establishes opportunities for ecological
ecological tradeoffs associated with performance in
tradeoffs in which traits that are beneficial in one envi-
disease and non-disease environments. These
ronment may be detrimental in the other environment
pathogens may therefore have different competitive
(Sokurenko et al., 2006; Brown et al., 2012; Mikonranta
et al., 2012). As consequence, facultative pathogens
Received 12 May, 2017; Revised: 20 October, 2017
accepted 25 October, 2017. *For correspondence. E-mail tgplatt@ may share a number of general features associated with
ksu.edu; Tel. 1785 532 6280; Fax 1785 532 6653. the similarities in their life histories. The importance of
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V
 Evolutionary ecology of crown gall disease 17

rhizosphere exudates

catabolized rhizosphere exudates

opines

catabolized opines

oncogene products

Agrobacterium cells

other microbes
Disease
environment
Non-disease
environment

Fig. 1. Facultative pathogens experience disparate ecological factors and evolutionary pressures associated with disease and non-disease
environments. Transitions between these environments (double-headed curved arrows) are critically important to the ecological and evolution-
ary dynamics of these pathogens. In the case of Agrobacterium tumefaciens (white ovals), non-disease environments include soil reservoirs
and uninfected host rhizospheres, both of which vary dramatically from the environment of diseased plant rhizospheres or actively infected
galls (light brown mass in upper root system). Each environment is associated with a unique microbiome (multicolored ovals) allowing for key
differences in the positive and negative microbial interactions that A. tumefaciens experiences in these environments. In addition, the need to
colonize plant surfaces (e.g., via the unipolar polysaccharide or UPP, red in inset) and antagonism from plant defenses are key features of
host plant environments. Within host environments, important host-microbe interactions also include the utilization of rhizosphere exudates,
including those (filled yellow circles) whose catabolism (curved blue arrow pointing to split yellow circles that represent catabolized rhizosphere
exudates) is conferred by the At plasmid (blue open circle), and pathogenesis conferred by the Ti plasmid (open circle with purple, orange and
grey regions). The T-DNA (purple region) found on the Ti plasmid is delivered via a type IV secretion system (gray cylinder) encoded by the
vir-region (gray region) of the Ti plasmid into the host plant cell (arrow through gray cylinder). This allows for T-DNA insertion into the plant
genome stimulating tumorigenesis via expression of oncogene products (filled yellow triangles) and opine (filled purple circles) production
(curved arrows originating at integrated T-DNA). Opine uptake and catabolism functions (curved orange arrow pointing to split purple circles
that represent catabolized opines) are conferred by the opine catabolic region (orange) of the Ti plasmid. Within the disease environment, the
pathogen also faces potential exploitative competition over opines and other plant exudates from other microbes able to use these resources
(e.g., arrow representing opine uptake by blue genotype) as well as potential interference competition (dashed, blunt line between white and
blue genotypes).

the transitions between environmental and host environ-
ments largely depends on tight regulatory control of key
attributes including virulence functions, motility and sur-
face colonization phenotypes and is shaped by interac-
tions with host defenses and features of the host
environment, including the host’s microbiome.
The generalist plant pathogen Agrobacterium tumefa-
ciens, the causative agent of the neoplastic plant disease
crown gall, has been an important model bacterium yield-
ing key insights into host-microbe signalling (Venturi and
Fuqua, 2013), bacterial cell-to-cell communication (Lang
and Faure, 2014) and virulence mechanisms (Christie
et al., 2005; Nester, 2015). Further, A. tumefaciens is a
prominent biotechnology tool because of its ability to
drive horizontal gene transfer to plants (Hwang et al.,
2015b) and an economically important pathogen of sev-
eral plant varieties (Pulawska, 2010). A. tumefaciens
pathogenesis and intraspecific interactions have been
studied extensively (Platt et al., 2014) and the molecular
processes underlying crown gall disease are established
(Fig. 1). In this review, we examine how this foundational
knowledge facilitates the use of A. tumefaciens as a valu-
able model system for studying the ecological and
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evolutionary dynamics of diseases caused by facultative
pathogens. We will focus our discussion on factors
related to the transition between disease and non-
disease environments as this defines a key challenge
shared by all facultative pathogens. In particular, we will
use agrobacterial pathogens as case studies to discuss
the importance and consequences of motility and surface
colonization phenotypes, interactions with hosts and
within host environments and the consequences of eco-
logical tradeoffs associated with the facultative pathogen
life history.
Ecology shapes pathogen diversification
Agrobacterial diversity
Pathogenic agrobacteria are a highly diverse polyphy-
letic group that includes the A. tumefaciens species
complex (formerly biovar 1), A. rhizogenes (formerly bio-
var 2) and A. vitis (formerly biovar 3) (Otten et al.,
2008). In each of these groups, a virulence plasmid hori-
zontally transferable to other bacteria confers most path-
ogenesis functions (Platt et al., 2014). Because this
complicates taxonomic classification, the pathogenic
18 I. S. Barton, C. Fuqua and T. G. Platt
agrobacteria and related bacteria have been the subject
of ongoing taxonomic revision (Farrand et al., 2003;
Young et al., 2003; Ormen ~o-Orrillo et al., 2015). For con-
venience, we will use a commonly used classification
system based on how these microbes interact with plant
hosts. Each group of pathogenic agrobacteria either
elicit a different pathogenesis phenotype in plants or
vary in their plant host range. A. tumefaciens causes
crown gall disease and can infect most dicotyledonous
plants. A. vitis pathogenesis also causes gall formation
but its host range is restricted to grapes and it addition-
ally triggers necrosis of grape roots. A. rhizogenes does
not cause gall formation, instead causing hairy root dis-
ease (Escobar and Dandekar, 2003).
Of these, the majority of research has centred on A.
tumefaciens and thus the discussion in this review will
focus on this taxon, often incorporating discussion of rel-
evant information about other agrobacteria. A. tumefa-
ciens possesses a multi-partite genome, generally
consisting of a circular chromosome (> 2 Mb) and
another large (> 2 Mb) chromid replicon (Slater et al.,
2009; Harrison et al., 2010). The chromid of strains
belonging to the A. tumefaciens species complex are lin-
ear molecules, while the chromids of A. rhizogenes and
A. vitis are circular molecules (Slater et al., 2013;
Ramı́rez-Bahena et al., 2014). The genome may also
include one or more plasmids (Goodner et al., 2001;
Wood et al., 2001; Huang et al., 2015). The tumor-
inducing or Ti plasmid (approximately 200 kb and 4% of
the genome) encodes most virulence functions and
imparts the ability to initiate crown gall disease. The sim-
ilarly sized root-inducing or Ri plasmids found in A. rhi-
zogenes strains correspondingly encode most functions
associated with hairy root disease. The Ti and Ri plas-
mids are similar in many aspects of their plasmid biology
and the mechanisms through which they confer the abil-
ity to infect host plants (Suzuki et al., 2009; Platt et al.,
2014). The regulation and mechanisms of agrobacteria
pathogenesis are well studied and have been the topic
of several recent review articles (Gelvin, 2012; Subra-
moni et al., 2014).
Ti and Ri plasmids are each highly diverse with a
mosaic structure containing several clusters of high con-
servation interspersed with more divergent sequence,
reflecting an evolutionary history marked by significant
horizontal gene transfer, deletions and other large-scale
genetic events (Otten et al., 2008; Suzuki et al., 2009).
Reflecting this plasmid diversity, pathogenic agrobacteria
are organized into groups based on variations associ-
ated with their Ti or Ri plasmids. Agrobacterial infection
causes plants to produce a specific set of opines that
varies depending on the particular Ti or Ri plasmid
(Fig. 1). Accordingly, Ti plasmids have been organized
into at least 10 groups based on the sets of opines they
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V
stimulate plants to produce during infection (Dessaux
et al., 1998). Further illustrating the diversity of Ti and Ri
plasmids is the observation that they belong to at least
four incompatibility groups—IncRh1, IncRh2, IncRh3
and IncRh4 (Otten et al., 2008). Plasmid incompatibility
groups are defined as sets of plasmids that are not sta-
bly inherited together when they co-occur within the
same genome. In the case of Ti and Ri plasmids, incom-
patibility reflects significant similarities in the repABC
region of the plasmid which encodes functions associ-
ated with the replication and partitioning of the plasmid
(Pinto et al., 2012). Remarkably, one extreme example
of incompatibility is a Ti plasmid that has two repABC
regions and is incompatible with both IncRh1 and
IncRh2 plasmids (Yamamoto et al., 2017).
The genomes of A. tumefaciens often include another
megaplasmid designated the At plasmid (often over 500
kb; 10% of the genome). The functions conferred by
these plasmids are less well defined, but they are known
to impart a number of metabolic activities associated
with rhizosphere environments thereby facilitating plant
colonization (Baek et al., 2005; Chai et al., 2007; Hau-
decoeur et al., 2009; Morton et al., 2014). Consistent
with this, pathogenic agrobacteria bearing both the Ti
and At plasmids outcompete cells bearing only the Ti
plasmid in the rhizosphere of actively infected hosts; in
contrast, they decline in frequency following plant senes-
cence (Morton et al., 2014). Like the Ti plasmid, the At
plasmid also exhibits dramatic variation across A. tume-
faciens strains.
Agrobacterial diversity is, in part, shaped by selective
pressures imposed by host plant environments. Reflect-
ing this, some A. tumefaciens and A. rhizogenes strains
exhibit relatively narrow host ranges and harbour Ti
plasmids larger than and lacking T-DNA region homol-
ogy with the majority of described Ti plasmids (Unger
et al., 1985). Examination of different strains revealed
that large scale genomic events (e.g., deletions associ-
ated with transposition events) can result in dramatic
narrowing of Ti plasmid plant host range, allowing for
the rapid emergence of variants with limited host range
(Paulus et al., 1991; van Nuenen et al., 1993). Another
example illustrating the impact of adaptation to host
environments on agrobacterial diversity is found with
many strains of A. vitis harboring a separate tartrate uti-
lization plasmid that confers a within host benefit stem-
ming from the ability to catabolize tartrate, a nutrient
present at high concentrations on their grapevine hosts
(Szegedi et al., 1992; Salomone et al., 1998; Burr and
Otten, 1999). Further, strains belonging to the genomo-
var G8 within the A. tumefaciens species complex carry
a number of genes specific to this group. Strain C58 is
within genomovar G8 and genome analysis reveals that
these specific genes are located within seven genomic

islands and include functions related to interaction with
plants such as the ability to catabolize plant exudates
and plant degradation products (Lassalle et al., 2011).
Transitions between disease and non-disease
environments
Motility, chemotaxis and vir-induction
Like other facultative pathogens, agrobacteria experi-
ence a wide range of environments. These include soil
reservoirs, the rhizosphere of plants not manifesting
crown gall disease and the rhizosphere of plants with
crown gall disease. For simplicity, we will refer to bulk
soil and uninfected plant rhizosphere environments as
non-disease environments and the rhizospheres of
infected plants as disease environments. Motility and
chemotaxis via flagellar locomotion is important for the
early steps of host colonization and virulence in A.
tumefaciens (Shaw et al., 1988; Chesnokova et al.,
1997). A. tumefaciens encodes chemotaxis receptors for
a variety of monosaccharides and sugar acids, many of
which that are exuded from plant host cells (Wright
et al., 1998). Several Ti plasmids, and the pAtK84b
opine catabolic plasmid carried by the avirulent A. rhizo-
genes strain K84, encode functions for chemotaxis
toward some of the opines that they confer the ability to
catabolize (Kim and Farrand, 1998). Plant produced
phenolics (e.g., acetosyringone and catechol) activate Ti
plasmid virulence (vir) gene expression through the
VirA-VirG two component system, and also stimulates
chemotaxis towards plant tissues (Bolton et al., 1986;
Shaw et al., 1988). A periplasmic binding protein, ChvE,
interacts with plant-released sugars and sugar acids and
potentiates vir-gene expression through VirA-VirG (He
et al., 2009; Hu et al., 2013). Acidic conditions also
enhance vir-gene expression partly through another
periplasmic regulator, ExoR, which is proteolytically
destabilized at low pH, thereby releasing the ChvG-ChvI
two component system to activate virG expression
(Heckel et al., 2014). Via these mechanisms, multiple
signaling pathways orchestrate virulence initiation
through finely coordinated virulence gene expression
upon recognition of host-associated signals.
Attachment and biofilm formation
Following motility and chemotaxis-mediated association
with host tissues, many pathogens adhere to and form
biofilms on host tissues in order to initiate virulence
functions. Initial surface contact can be mediated by pili
as found in a broad range of pathogens (Strom and
Lory, 1993; Craig et al., 2004; Craig and Li, 2008). A.
tumefaciens contains a gene cluster encoding Type IVb
pili (Ctp pili) that promote weak, reversible surface
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Evolutionary ecology of crown gall disease 19
interactions (Wang et al., 2014). Homologous loci found
in other bacteria are also thought to encode pili that
drive initial, non-specific attachment to surfaces
(Kachlany et al., 2000; Goodner et al., 2001; Tomich
et al., 2007). Reversible surface association via the Ctp
pili in A. tumefaciens likely facilitates the transition into
permanent attachment and biofilm maturation through
production of exopolysaccharides, most notably a polar
adhesin called the unipolar polysaccharide (UPP), which
is deployed upon contact with biotic and abiotic surfaces
(Fig. 1; Tomlinson and Fuqua, 2009; Li et al., 2012; Xu
et al., 2012). A. tumefaciens produces a number of other
exopolysaccharides, but only the UPP and cellulose
have been proposed to play a direct role in adhesion
(Matthysse, 1983; Matthysse et al., 2005). Other poly-
saccharides, including succinoglycan, cyclic b-1,2-
glucans and curdlan have variable and inconsistent roles
in attachment and biofilm formation (Heindl et al., 2014).
Regulation of attachment in A. tumefaciens is affected
by environmental conditions such phosphorus limitation,
oxygen availability, low pH and divalent cations such as
Fe and Mn (Danhorn et al., 2004; Ramey et al., 2004;
Tomlinson et al., 2010; Xu et al., 2012; Xu et al., 2013;
Heckel et al., 2014; Heindl et al., 2014; Feirer et al.,
2015). Many of these signals appear to modulate UPP
and cellulose production via effects on the bacterial sec-
ond messenger cyclic diguanylate monophosphate
(cdGMP) (Amikam and Benziman, 1989; Barnhart et al.,
2013; Xu et al., 2013; Barnhart et al., 2014; Feirer et al.,
2015). To what extent these factors modulate attach-
ment during pathogenesis of the host remains poorly
understood.
Navigating the host and its defenses
Host physiology and defenses are key aspects of the
within host environment of any pathogen. Plants often
activate innate immune responses when their pattern
recognition receptors (PRRs) bind microbe- or pathogen
associated molecular patterns (MAMPS or PAMPS),
which are associated with highly conserved microbial
features such as flagellin, elongation factor Tu, peptido-
glycan and lipopolysaccharides (Newman et al., 2013).
Though flagellin is one of the most common PAMPs that
plants respond to, the Arabidopsis flagellin PRR fails to
elicit a response to flagellin from Agrobacterium (Bauer
et al., 2001). However, Arabidopsis does mount a
defense in response to peptidoglycan from Agrobacte-
rium (Erbs et al., 2008) and when its elongation factor
PRR detects Agrobacterium elongation factor (Zipfel
et al., 2006).
In contrast to many phytopathogens, most plant hosts
fail to activate a hypersensitive response (HR) to A.
tumefaciens. This is thought to reflect the fact that A.
20 I. S. Barton, C. Fuqua and T. G. Platt
tumefaciens employs several mechanisms to interfere
with host defense systems. For example, A. tumefaciens
produces a catalase that prevents H2O2 accumulation
during the early stages of pathogenesis of Arabidopsis
thaliana, thereby interfering with HR and other host
defensive responses (Xu and Pan, 2000; Lee et al.,
2009). The phenolic inducers that activate the VirA-VirG
two component system coordinating the expression of
pathogenesis functions are themselves toxic and may
thereby contribute to the plant’s defense against agro-
bacteria. However, VirH2 expressed following vir-induc-
tion detoxifies most phenolic inducers and even allows
for the catabolism of some of these phenolics (Brencic
et al., 2004).
Much of the work in this area has focused on interac-
tions between A. thaliana plants and a generally narrow
subset of A. tumefaciens taxa. Future work should eval-
uate how well these interactions represent those occur-
ring on other hosts and with other agrobacteria. For a
more thorough discussion of the interactions between
A. tumefaciens and plant defenses, as well as how
A. tumefaciens impacts hosts tissues during the devel-
opment of crown gall disease, we recommend two
recent review papers (Gohlke and Deeken, 2014; Hwang
et al., 2015a).
Consequences of infection—virulence plasmid
conjugation
The complex regulatory network underlying the horizon-
tal genetic transfer (HGT) of Ti plasmids involves
responses to host cues and quorum sensing signalling
(Lang and Faure, 2014). Following transformation of
plant cells with T-DNA (Fig. 1, Gelvin, 2012), the result-
ing release of opines from the infected plant tissue is
perceived through transcription activators (e.g., AccR,
NocR or OccR) that specifically form complexes with
specific opines, and initiate gene expression for catabo-
lism of that specific opine (Fig. 1; von Lintig et al.,
1991; von Lintig et al., 1994; Kim and Farrand, 1997;
Subramoni et al., 2014).
A subset of opines, the so-called conjugative opines,
also lead to elevated expression of traR, the primary
activator of Ti plasmid conjugative transfer between bac-
teria (White and Winans, 2007). TraR is a LuxR-type
quorum sensing regulator that recognizes the acylated
homoserine lactone (AHL) quorum-sensing molecule,
N-(3-oxooctanoyl)-L-homoserine lactone (3OC8-HSL),
synthesized by another Ti plasmid gene product called
TraI, a LuxI-type AHL synthase. TraR-AHL complexes
promote transcription of the conjugative transfer genes
for the Ti plasmid, tra/trb, including the traI gene,
thereby creating a positive feedback induction loop
(Piper et al., 1993; Zhang et al., 1993; Fuqua and
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Winans, 1994; Hwang et al., 1994). TraR-AHL com-
plexes also increase Ti plasmid copy number through
activation of the repABC genes (Fuqua and Winans,
1996; Pappas and Winans, 2003; White and Winans,
2007; Pinto et al., 2012). Quorum-dependent regulation
of conjugation is further controlled through an antiactiva-
tor, TraM, which directly inhibits activated TraR (Hwang
et al., 1999). In some cases non-conjugative opines can
activate expression of defective, truncated TraR
paralogues (called TraS or TrlR) which also can inhibit
activation of conjugative transfer genes (Oger et al.,
1998; Zhu and Winans, 1998).
These complex gene regulatory mechanisms ensure
that Ti plasmid conjugation rates and copy number
increase in response to opine availability and the density
of the agrobacterial population. This intricate control
reflects the complex evolutionary dynamics at work in
controlling the Ti plasmid copy number and HGT. Experi-
mental evolution of a strain that constitutively expresses
the quorum sensing regulon under lab conditions results
in the spread of mutants that minimize the costs of
expressing quorum sensing regulated functions (Tan-
nieres et al., 2017). This result illustrates that there are
significant costs associated with expression of these
systems. The availability of opines likely counterbalan-
ces Ti plasmid costs, including those associated with
conjugation and provides a fitness benefit to harbouring
the otherwise costly plasmid. These conjugation events
may result in novel plasmid-host background combina-
tions when the Ti plasmid is delivered to plasmidless
agrobacteria. Alternatively, Ti plasmids may be delivered
into genotypes that already contain a Ti plasmid. In the
case of similar Ti plasmids, a quorum sensing regulated
entry exclusion system may reduce the efficiency of
these events (Cho et al., 2009). However, when conju-
gation does result in co-resident Ti plasmids there are
several possible outcomes that are potentially significant
to the evolution of Ti plasmids. First, the co-resident
plasmids may separate due to incompatibility which
could result in novel plasmid-host background combina-
tions (Cho et al., 2009). Second, the plasmids may
exchange genetic regions or co-integrate via homolo-
gous recombination. Such events, coupled with subse-
quent transposition and large scale deletion events may
help account for the chimeric nature and high degree of
diversity exhibited by Ti and Ri plasmids (Otten et al.,
1992; Otten et al., 2008).
Additionally, other host factors may modulate HGT of
virulence plasmid transmission in A. tumefaciens. Plant-
produced c-amino butyric acid (GABA) accumulates in
acidic wound conditions (Fig. 1) and activates the At
plasmid blcABC operon after being taken up through the
proline/GABA receptor and ABC-transporter (atu2422
and braE (atu2427), respectively) and converted to
 Evolutionary ecology of crown gall disease 21

DISEASE PROGRESSION

Fig. 2. Facultative pathogens often infect hosts from environmental populations or reservoirs (leftmost curved arrow). As a consequence, path-
ogenesis results in a dramatic shift in the pathogen’s biotic and abiotic environment. In this highly simplified schematic, each colour represents
a different microbial genotype. These may be resident microbiota, microbes that subsequently colonize the host or novel genetic variants aris-
ing via genetic changes (e.g., mutation or horizontal gene transfer). The space defined by the solid curvy lines represents the within-host envi-
ronment, while the space outside this represents the pathogen’s environmental reservoir. Dynamic selective pressures stemming from changes
in the competitive environment, environmental conditions, and host responses can result in shifts within host-associated microbial populations
and communities as the disease progresses (arrows exclusively inside the within-host environment). At any point over the course of the infec-
tion there is potential for shedding of the host-associated microbiota into the environmental reservoir (curved arrows exiting the within-host
environment).

semi-salicylic acid (SSA) (Chevrot et al., 2006). Salicylic
acid (SA), a plant defense molecule, has also been
shown to reduce vir expression and activates expression
of blcABC genes (Yuan et al., 2007; 2008). BlcC is a
c-butyrolactonase that is capable of degrading the
3OC8-HSL and is proposed to be involved in quorum
quenching (Khan and Farrand, 2009; Haudecoeur and
Faure, 2010; Lang and Faure, 2014). Other components
in the pathway from c-butyrolactone (GBL) to succinate,
including GBL, c-hydroxybutyrate (GHB) and semi-
salicylic acid (SSA) also increase blcABC gene expres-
sion (Haudecoeur and Faure, 2010; Lang and Faure,
2014; Subramoni et al., 2014). It is possible that quorum
quenching in A. tumefaciens may provide a competitive
advantage. In addition to enabling metabolism of plant-
release GBL for energy, this system would generally
reduce the energy expenditure due to quorum signalling
by A. tumefaciens and other proximal bacteria, (Chevrot
et al., 2006; Chai et al., 2007; Yuan et al., 2007;
Subramoni et al., 2014), although there has been some
disagreement about on the strength of these effects
(Khan and Farrand, 2009).
Consequences of ecological tradeoffs
Eliciting host pathogenesis is a costly behavior that can
greatly reduce a pathogen’s cellular growth rates and fit-
ness (Ackermann et al., 2008; Platt et al., 2012a; Mor-
ton et al., 2014; Peyraud et al., 2016). Each episode of
host infection results in a dramatic shift in the patho-
gen’s biotic and abiotic environment. This requires a
physiological reprogramming, shapes selective pres-
sures acting on pathogen populations and can alter the
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microbial community dynamics (Fig. 2). As an example
of this, the endophytic bacterial community of grapevine
galls caused by A. vitis is distinct from the community
found within analogous healthy tissues, suggesting that
opine availability results in a shift in the bacterial com-
munity (Faist et al., 2016). Pathogens must deal with
host defenses as well and inter- and intra-specific micro-
bial competition, the burden of which can severely limit
metabolic potential. Numerous strategies have evolved
to promote the maintenance of burdensome pathogenic
functions across the wide variety of niches that faculta-
tive pathogens occupy.
Tradeoff between growth and pathogenesis
The expression of virulence functions is often very costly
leading to reduced vegetative growth rates, thereby
establishing a tradeoff associated with pathogenesis
(Maharjan et al., 2013; Ferenci, 2016; Peyraud et al.,
2016). Many pathogens limit these costs by tightly regu-
lating the expression of pathogenesis functions, thereby
minimizing costs and promoting the evolutionary mainte-
nance of these traits. It is common for pathogens to reg-
ulate virulence functions in a density-dependent manner,
assuring a sufficient number of pathogenic bacteria to
cause disease (Gama et al., 2012). The plant pathogen
Ralstonia solanacearum coordinates virulence gene
expression through a quorum-dependent global regula-
tor, phcA (Peyraud et al., 2016). Expression of phcA has
been shown to reduce fitness and metabolic potential,
but it is essential for successful invasion of host xylem
tissue (Peyraud et al., 2016). Thus for this and other
systems there is a selective pressure to curtail expres-
sion of costly virulence functions, but through the
22 I. S. Barton, C. Fuqua and T. G. Platt
coordination afforded in a quorum-regulated system, the
majority of cells in the population concertedly participate
in the behaviour.
Other pathogens, such as Salmonella typhimurium,
initiate virulence functions through phenotypic noise,
where a small, isogenic population, to their own detri-
ment, stochastically expresses costly, virulence behav-
iours that benefit a surrounding infecting population
(Ackermann et al., 2008). In this way, a subset of the
population can incur costs of virulence to the benefit of
a faster-growing subpopulation that does not activate vir-
ulence (Ackermann et al., 2008; Diard et al., 2013).
While some of the individuals incur the often heavy bur-
den of virulence initiation, the larger population is mostly
unaffected and can initiate subsequent virulence expres-
sion because of retention of unexpressed virulence
traits.
In A. tumefaciens, virulence induction significantly
reduces fitness by dramatically decreasing population
growth rates (Platt et al., 2012b). The VirA-VirG two
component system accordingly limits this costly expres-
sion to the host environment based on a range of plant
rhizosphere cues, including phenolic compounds, spe-
cific sugars, acidic conditions and low phosphorus levels
(Venturi and Fuqua, 2013). Further, the initial conse-
quences of virulence induction are seemingly offset by
the availability and metabolic access to opines that are
produced as a result of successful infection (Guyon
et al., 1993; Platt et al., 2012a). However, the heavy
cost of vir-induction establishes a strong selective pres-
sure favouring the spread of avirulent mutants (Platt
et al., 2012a). This likely accounts for several observa-
tions of mutants unable to infect hosts spreading under
in vitro vir-inducing conditions (Fortin et al., 1992; 1993)
and in planta (Belanger et al., 1995; Llop et al., 2009).
The results of Llop et al. (2009) suggest that these
mutants more often colonize the diseased host from
environmental reservoirs rather than arising from de
novo mutation in the infecting strain.
In addition to the large cost associated with expres-
sion of virulence genes, there is also a significant but
comparatively smaller carriage cost to harbouring a Ti
plasmid when carbon or nitrogen are limiting resources
(Platt et al., 2012b). Because of this, non-disease envi-
ronments, wherein opines are absent, are predicted to
be a sink due to competition from saprophytic agrobac-
teria or other competitors (Platt et al., 2012a). For exam-
ple, Krimi et al. (2002) observed a decrease in the
frequency of pathogenic genotypes within the agrobacte-
rial population associated with the absence of infected
plants. Experimental support for this idea comes from
the observation that pathogenic agrobacteria are rapidly
outcompeted by non-pathogenic agrobacteria when
coresident in a rhizosphere without opines. Conversely,
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V
pathogenic genotypes outcompete the non-pathogenic
agrobacteria when competing in a rhizosphere into
which opines are excreted (Guyon et al., 1993). The
consequences of non-disease environments acting as a
sink for pathogenic agrobacteria likely has a profound
effect on the epidemiology of crown gall disease.
Competition within plant microbiomes
Once pathogens stably colonize a host, interference
competition between the pathogen and members of the
microbiome can affect the ability of the pathogen to
maintain its host-associated niche (Figs 1 and 2; Ghoul
and Mitri, 2016). Interference competition can be medi-
ated by antagonistic behaviours such as Type VI secre-
tion systems (T6SSs) and production of antimicrobials
(Massey et al., 2004; Basler et al., 2013; Kapitein and
Mogk, 2013; Ghoul and Mitri, 2016). Initiation of compet-
itor killing may be beneficial during initial host coloniza-
tion to compensate for decreased fitness due to
expression of costly virulence functions. It is likely impor-
tant for A. tumefaciens to stabilize its niche in the face
of intense competition among rhizosphere microbiota.
Among these mechanisms, A. tumefaciens activates a
T6SS under acidic conditions through the ChvG-ChvI
two component system (Wu et al., 2012; Heckel et al.,
2014). Interestingly, pairwise competition with the oppor-
tunistic human pathogen Pseudomonas aeruginosa
hinges on the A. tumefaciens T6SS. The A. tumefaciens
T6SS invokes lethal retaliation by P. aeruginosa (via its
own T6SSs) under laboratory conditions, but results in
the dominance of A. tumefaciens over P. aeruginosa in
planta (Ma et al., 2014). Other factors also influence this
interaction, such as production of diffusible molecules
that can inhibit growth or disperse established A. tume-
faciens biofilms (Barreteau et al., 2009; Hibbing and
Fuqua, 2012; Ghequire et al., 2017).
In some cases, agrobacterial infection may indirectly
result in host environment conditions that are unfavoura-
ble to other microbes. For example, tomato expression
of the A. rhizogenes oncogene rolB results in both stim-
ulation of meristem formation associated with the pro-
duction of adventitious, ‘hairy’ roots (Britton et al., 2008)
and increased resistance to the foliar fungal pathogens
Alternaria solani and Fusarium oxysporum (Arshad
et al., 2014). Pathogens may also outcompete rival
microbes in host environments via exploitive competi-
tion, wherein resource consumption limits the availability
of that resource for its competitors (Figs 1 and 2;
Ghoul and Mitri, 2016). For example, production of side-
rophores could increase competitiveness in low iron
environments, and increased motility and nutrient
sequestration could limit nutrient availability to competi-
tors (Ghoul and Mitri, 2016; Niehus et al., 2017).

A. tumefaciens engineers a competitive nutritional advan-
tage during plant infection through the production of
opines (Fig. 1), the catabolism of which is also predomi-
nantly encoded on the Ti plasmid (Kim et al., 2008; Platt
et al., 2012a). Strains of a few other species of rhizo-
sphere bacteria are also able to catabolize opines (Guyon
et al., 1993; Moore et al., 1997; Brencic and Winans,
2005; Farrand et al., 2007). Consequently, pathogenic
agrobacteria face interspecific competition for the utiliza-
tion of opine resources within the disease environment. In
addition, pathogenic agrobacteria compete for access to
opines with other agrobacteria that harbour different Ti
plasmids or other plasmids conferring the ability to catabo-
lize opines (Lang et al., 2017). The biocontrol agent A. rhi-
zogenes K84 provides a particularly dramatic example.
This avirulent, opine catabolic strain produces several
antimicrobials that target virulent nopaline type A. tumefa-
ciens and A. rhizogenes strains (Penyalver et al., 2001;
Kim et al., 2006; Platt et al., 2014).
A number of other potential biocontrol strains influenc-
ing the establishment of crown gall disease have been
identified. For example, A. vitis F2/5 can inhibit the
establishment of crown gall disease in grapevine hosts,
but fails to do so on other host plants such as tobacco
(Kaewnum et al., 2013). This strain is non-tumorigenic
but causes necrosis of grape tissues (Zheng and Burr,
2016) and, similar to the biocontrol agent A. rhizogenes
K84, A. vitis F2/5 carries an opine catabolic plasmid
(Szegedi et al., 1999). However unlike K84, F2/5 does
not appear to produce a bacteriocin antagonizing the
growth of tumorigenic A. vitis strains (Burr et al., 1997).
The mechanism of F2/5’s grape tumor inhibition is not
fully characterized, however, appears to depend on this
strain producing a nonribosomal peptide synthetase
product that interferes with host transformation by tumor-
igenic A. vitis strains (Kaewnum et al., 2013; Zheng and
Burr, 2016). In contrast, the mechanism of inhibition by
other potential biocontrol agents of agrobacterial patho-
gens does appear to involve the production of bacterio-
cins. These include A. vitis E26 (Wei et al., 2009) and
A. vitis VAR03-1, both of which produce bacteriocins
that inhibit A. tumefaciens, A. rhizogenes and A. vitis
strains (Kawaguchi et al., 2008; Li et al., 2009).
Several non-agrobacterial rhizobacteria are also
potential biocontrol agents inhibiting the establishment of
crown gall disease. For example, inoculation of roots
with Bacillus subtilis BSCH14 prior to planting in the
field resulted in significant inhibition of crown gall dis-
ease incidence (Rhouma et al., 2008). Further, another
study identified that a number of different rhizobacteria,
including Pseudomonas putida UW4 and Burkholderia
phytofirmans PsJN, that produce the enzyme 1-
aminocyclopropane-1-carboxylate deaminase (ACCD)
antagonize crown gall disease development on tomato
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V
Evolutionary ecology of crown gall disease 23
plant hosts by both A. tumefaciens Sh-1 and A. vitis S4
(Toklikishvili et al., 2010).
Pathogenic agrobacteria compete with several other
microbes over host produced opines. Other bacterial
and even fungal taxa have also evolved the ability to uti-
lize opines in addition to related agrobacteria and rhizo-
bia (Beauchamp et al., 1990; Bell et al., 1990; Bergeron
et al., 1990). Therefore, although opines are custom
nutrients produced almost exclusively as a result of
infection by pathogenic agrobacteria, they are not an
exclusive private good. The degree to which opine utili-
zation influences the competitiveness of A. tumefaciens
during infection and whether spatial or other factors are
important for niche stabilization remains to be experi-
mentally addressed.
Selection for avirulence—resisting invasion by cheaters
through HGT
Pathogenesis initiates a shift in the selective pressures
acting on pathogens and can influence the ecological
dynamics of infecting populations (Fig. 2). Especially in
cooperative systems, there exist selective pressures that
act to disfavour organisms from participating in viru-
lence, resulting in a rise of cheaters with diminished vir-
ulence phenotypes that can outcompete the virulent
progenitor (Gama et al., 2012). For example, loss of
cooperative siderophore production occurring during
chronic Pseudomonas infections has been attributed to
the competitive social interactions fostered within the
host environment (Andersen et al., 2015). Similarly, avir-
ulent variants that arise during S. typhimurium infections
have a selective advantage during host colonization due
to the loss of costly expression of virulence functions
and cooperation (Ackermann et al., 2008; Diard et al.,
2013). In the face of these negative selective pressures,
cooperative traits must be supported or reinforced to
allow for persistence across generations and subse-
quent infection cycles.
Cooperative pathogenesis genes can be stabilized by
the dissemination of pathogenicity genes via HGT
(smith, 2001; Dimitriu et al., 2014). There are numerous
mechanisms by which bacterial pathogens can horizon-
tally acquire novel genetic material, including conjuga-
tion, transformation, transduction and gene transfer
agents (GTAs). Among these mechanisms, conjugation
is arguably the most common and a stable mechanism
to faithfully regulate and coordinate dissemination of vir-
ulence genes (Cabezon et al., 2015; von Wintersdorff
et al., 2016). Conjugative elements that contain pathoge-
nicity functions include integrative and conjugative ele-
ments (ICEs) and conjugative plasmids. ICEs were
initially discovered due to their ability to confer resis-
tance to antibiotics and heavy metals, but pathogenicity
24 I. S. Barton, C. Fuqua and T. G. Platt
and symbiotic functions are also frequently contained
within these elements (Johnson and Grossman, 2015).
For example, Pseudomonas aeruginosa contains a path-
ogenicity island, PAPI-1, that also encodes its own con-
jugative machinery (Carter et al., 2010). Similarly, the
symbiotic island, ICEMlSymR7a allows members of the
genus Mesorhizobium to form nodules on Lotus species
and is self-conjugative (Ramsay et al., 2006).
More commonly pathogenicity functions are carried on
self-transmissible plasmids that contain numerous viru-
lence genes as well as maintenance and replication
functions, and these elements can be quite large (Sen-
gupta and Austin, 2011). Very tight regulation of copy
number, gene expression and plasmid maintenance lim-
its the cellular burden of plasmids, as well as promoting
proper segregation and dissemination. In several cases,
unstable plasmid maintenance may be compensated by
increased interspecies HGT, as seen in recent experi-
ments in Pseudomonas (Hall et al., 2016). In A. tumefa-
ciens, coordination of HGT with the availability of opines
and the density of the virulent population (via quorum
sensing) may provide a means to ensure delivery and
stabilization of the plasmid in avirulent cells that colonize
or arise via mutation within the disease environment.
Dissemination of the Ti plasmid in the presence of
opines may also stabilize the Ti plasmid by allowing the
plasmid to colonize genetic backgrounds that can out-
compete the infecting strain. Opine-derived fitness bene-
fits are thought to be critical for the persistence of
cooperation and stabilization of virulence in A. tumefa-
ciens populations (Platt et al., 2012a). However,
because of the diversity in plasmid types and the impor-
tance of spatial and temporal variation, the potential
value of Ti plasmid conjugation in promoting the persis-
tence of pathogenic agrobacteria within natural disease
environments remains elusive.
Concluding remarks
The plant pathogen A. tumefaciens has proven a valu-
able system for the study of host-microbe and intraspe-
cific microbe-microbe interactions (reviewed by Venturi
and Fuqua, 2013; Kado, 2014; Lang and Faure, 2014;
Hwang et al., 2015a). Extensive work has also examined
key aspects of its virulence plasmid biology (Platt et al.,
2014) and colonization of host and non-host surfaces
(Heindl et al., 2014). In this review, we argue that the
biology of this pathogen and the foundation of mecha-
nistic understanding positions A. tumefaciens well as a
model for the ecology and evolution of infectious dis-
eases caused by facultative pathogens. An understand-
ing of the ecology and evolution of pathogens with this
life history necessarily requires inclusion of their dynam-
ics within and the linkages between populations residing
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V
within these disparate disease and non-disease environ-
ments. Towards that goal, future research should
address some current knowledge gaps related to how A.
tumefaciens behaves in its environmental reservoirs
(e.g., soil and commensal states in plant rhizospheres)
as well as how it transitions between disease and non-
disease environments via shedding and disease trans-
mission. This effort would be well served by a compre-
hensive sampling of agrobacteria diversity, blind to the
historical biases toward pathogenic agrobacteria found
associated with crown gall disease tissues. Another
opportunity for future research involves determining
whether A. tumefaciens undergoes significant pathoa-
daptation via within-host evolution, as has been docu-
mented in a number of pathogens that infect from non-
disease environments (Sokurenko et al., 1998; Marvig
et al., 2015). Interspecific interactions (e.g., Ma et al.,
2014) as well as competitive interactions with avirulent
agrobacteria (Kim et al., 2006) can have dramatic con-
sequences on the success of pathogenic agrobacteria
on host tissues. Despite this, the consequences of
crown gall disease on plant microbiomes has not been
well characterized nor is it known whether or not the
composition of the host microbiome significantly alters
the initiation or progression of crown gall disease.
Acknowledgements
Research in the area of agrobacterial disease was funded by a
National Institutes of Health grant (R01 GM092660; CF) and
currently by a Faculty Research Support Program grant (ISB
and CF) through Indiana University. Research on the microbial
interactions of agrobacteria was funded by a National Science
Foundation grant (MCB 1650187; TGP). The authors declare
that they have no conflict of interest associated with this work.
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